The development of "natural" immunosuppressive strategies to obviate T cell alloreactivity towards grafted tissues is a highly desirable alternative to non-specific, drug-mediated, T cell immunosuppression. We have shown, by gene transfer in a preclinical miniature swine kidney transplant model, that a partial donor/recipient match for a single MHC class II (CI2) molecule promotes spreading T cell tolerance to subsequent transgene-matched grafts and the down-regulation of host T cell activation to all graft-associated major and minor antigens. Given that 1) Partial CI2 matching also fosters regulatory tolerance to human transplants and 2) The miniature swine is the only animal model in which the role of CI2 in T cell tolerance can be reliably studied, the goal of this project is to elucidate, using the CI2 gene transfer pig model, the mechanism of regulatory T lymphocyte tolerance mediated by MHC CI2. This knowledge is a necessary prerequisite to the development of therapeutic protocols that activate the natural suppression of anti-graft responses in humans. Recent data using this approach indicate that CI2 transgenesis-mediated tolerance is associated with the down-regulation of T cell reactivity to the graft by regulatory T (T-reg) cells. In addition, transgenic CI2-derived peptides seem to be involved in the activation of T-reg cells which migrate to the graft. These findings suggest that Tg CI2 peptides fashion part of the host T-reg cell repertoire that may, subsequently, be activated by graft-matched CI2 to develop regulatory tolerance to the transplant. The present study intends to evaluate the importance of regulatory tolerance among other tolerogenic mechanisms leading to allograft acceptance. To this end, we will 1) Examine the effects of intracellular (peptides) and surface CI2 transgene expression on tolerance induction;2) Assess the effects of CI2 transgene expression patterns on host immunity to Tg-matched kidney grafts and 3) Characterize the specificity and mode of action of T-reg-mediated suppression. It is hoped that these studies will provide the impetus for the design of new therapeutic molecules with clinical application in transplantation biology.